A major synthetic value of enzyme catalysis is its usually predictable stereoselectivity. See, e.g., Whitesides et al., Angew. Chem. Int. Ed. Encl., 24:617 (1985); Jones, J. B. Tetrahedron, 42:3351 (1986); Yamada et al., Angew. Chem. Int. Ed. Engl., 27:622 (1988); Wong, C.-H., Science, 244:1145 (1989); Ohno et al., Org. React. 37:1 (1989); Chen et al., Angew. Chem. Int. Ed. Engl., 28:695 (1989).
A change of stereoselectivity, however, may occur, though very unusual, with different substrate structures, temperatures or solvents. See, e.g., Mohr et al., Helv. Chim. Acta, 66:2501 (1983); Sabbioni et al., J. Chem. Soc. Chem. Commun., 236 (1984); Ohno et al., J. Am. Chem. Soc. 103:2405 (1983); Wang et al., J. Org. Chem. 53:3127 (1988); Lalonde et al., J. Am. Chem. Soc. 103:2405 (1981); Wang et al., J. Org. Chem., 53:2323 (1988); Pham et al., J. Am. Chem. Soc., 111:1935 (1989); Keinan et al., J. Am. Chem. Soc., 108:162 (1986); Sakurai et al., J. Am. Chem. Soc., 110:7236 (1988); Fitzpatrick et al., J. Am. Chem. Soc. 113:3166 (1991). These selectivity changes are often not very significant, with some exceptions where the enantioselectivity is inverted.
In the case of enzymatic aldol reactions, the diastereofacial selectivity for the aldehyde component is often consistent and completely controlled by the enzyme as documented by numerous reactions catalyzed by fructose-1,6-diphosphate aldolase or N-acetylneuraminic acid (or sialic acid) aldolase (EC 4.1.3.3). In most cases, the "D" isomer of an .alpha.-substituted aldehyde reacts faster than the "L" isomer, both with si-facial selectivity. The Cram-Felkin mode of attack on the "D" aldehyde is therefore proposed for the transition state. See, e.g., Toone et al., Tetrahedron, 45:5365 (1989); Bednarski et al., J. Am. Chem. Soc., 111:627 (1989); Straub et al., J. Org. Chem., 55:3926 (1990); Durrwachter et al., J. Org. Chem., 53:4175 (1988); von der Osten et al., J. Am. Chem. Soc., 111:3924 (1989); Kajimoto et al., J. Am. Chem. Soc., 113:6187 (1991); Auge et al., New J. Chem., 12:733 (1988).
Because of the stereoselectivity of enzymes such as aldolases that participate in the metabolism of carbohydrates, it is extremely difficult to design and make new carbohydrates that can be used to study carbohydrate metabolism. There is a need for such synthetic compounds for use as experimental tools in elucidating the molecular character of the numerous and varied pathways involved in carbohydrate anabolism and catabolism.
Of particular relevance to the present invention is the sugar, N-acetylneuraminic acid (NeuAc) or sialic acid. NeuAc is an integral component of most cells and is believed to play a major role in imparting electrical charge characteristics to such cells. Further, NeuAc-like compounds such as the eight and nine-carbon sugar moieties KDO and KDN are major constituents of non-mammalian tissues.
N-Acetylneuraminic Acid (NeuAc) aldolase, also commonly referred to as sialic acid aldolase is a type I aldolase known to form an enamine intermediate with pyruvate, which reversibly reacts with the second substrate N-acetylmannosamine to give NeuAc. See, e.g., Deijl et al., Biochem. Biophys. Res. Commun., 111:668 (1983); and Shukla et al., Anal. Biochem., 158:158 (1986).
NeuAc aldolase is known to accept many aldoses as acceptor substrates. In all previously known aldol condensation reactions with such acceptor substrates, the eneamine intermediate approaches the si face of the incoming aldehyde substrate to form a new stereogenic center of S configuration. Anti-Cram-Felkin attack is generally observed for good chiral aldehyde substrates and Cram-Felkin attack is observed for weak substrates. In both cases, a si-facial selectivity was observed. See, e.g., Auge et al., New J. Chem., 12:733 (1988); and Auge et al., Tetrahedron, 46:201 (1990).
Based on such current knowledge concerning aldolase stereoselectivity, therefore, NeuAc aldolase is considered to be useful only for the production of D-sugars having S configuration. As is disclosed hereinafter, NeuAc aldolase has now unexpectedly been found to be capable of the production of certain L-sugars having a formed stereogenic center of R configuration.